Oxygen is absorbed through the lungs and carried by hemoglobin in red blood cells (RBC) for delivery to tissues throughout the body. At high oxygen concentrations, such as those found in the proximity of the lungs, oxygen binds to hemoglobin, but is released in areas of low oxygen concentration, where it is needed.
Each hemoglobin molecule in an adult consists of two .alpha.-globin subunits and two .beta.- or .delta.-globin subunits or chains. The globin chains themselves are synthesized from templates, called mRNAs which are, in turn, synthesized from a master template which is the globin gene. The gene contains three regions (exons) which code for the amino acids of the globin and contains other regions, called intervening sequences (IVS), that are transcribed, but then removed from the primary transcript when the mature mRNA is formed. The mature mRNA is then translated into the globin protein.
Each globin chain contains an iron atom coordinated by the two imidazole nitrogens of two histidines. The iron atom is surrounded by a protoporphyrin ring which is contained within the hemoglobin molecule but not chemically linked to it. The protoporphyrin and the iron atom together form a heme group which binds the oxygen molecule and of course, gives hemoglobin its name. Thus, each hemoglobin tetramer is capable of binding four molecules of oxygen. Hemoglobin that contains two .alpha.-globin chains and two .beta.-globin chains is called Hemoglobin A (HbA); the other form of adult hemoglobin, which has two .delta.-globin chains is termed HbA.sub.2. Both types of hemoglobins are synthesized in the same cells, called erythrocyte precursors, which, as they mature become recticulocytes, and finally become erythrocytes or red blood cells (RBC). Normally only about 2.0-2.5% of the hemoglobin will be HbA.sub.2, although persons having a genetically defective .beta.-globin gene are known to express as much as 15% HbA.sub.2. Schroeder, W. A., et al., 1975, BIOCHEM. GENET. 10:135; Steinberg, M. H., et al., 1982, J.LAB.CLIN.MED. 100:548. No hemoglobin has both a .beta. and a .delta. chain.
Many medical and surgical conditions require the administration of whole blood or of the RBC fraction of blood (packed RBC). At present the only sources of RBC suitable for clinical use are human donors. It is, however, expensive to procure human blood in the quantities required. Moreover, even when an adequate amount of blood is obtainable there are many risks associated with the transfusion of blood from one individual to another. The avoidance of these risks entails yet more expense and the efforts of highly trained medical practitioners, which, even though prodigious, are not completely successful. Transfusion reactions and blood-born infections remain a substantial and significant risk. The risks of transfusion and limited availability of blood deter physicians from using blood replacements in all but the most necessary cases.
Therefore, it would be highly desirable to be able to use manufactured hemoglobin as a blood substitute rather than obtain blood from human volunteers. Because the function of hemoglobin is entirely independent of the RBC, the hemoglobin derived from transgenic animals could be used as a blood substitute with only slight modifications such as cross-linking, Ogden, J. E., et al., 1992, BIOMATERIALS, ARTIFICAL CELLS & IMMOBILIZATION TECHNOLOGY 20: 473. The preferred type of hemoglobin for this purpose is HbA.sub.2. HbA.sub.2 is more stable than HbA, Kinderlerer, J., et al., 1970, BIOCHEM.J. 119:66P, which would allow for a longer life-span of the product and would also allow the use of harsher conditions during the manufacture of the synthetic product. Secondly, the physico-chemical differences between HbA.sub.2 and the HbAs of other animals are larger than between human HbA and the nonhuman HbAs. Therefore, the process of purifying the desired human Hb from other types of hemoglobins is significantly easier.
The efforts directed towards the production of human globin chains and HbA in transgenic animals have been considerable. See, e.g., Logan et al., WO92/22646, Holis et al., WO92/11380, and Grosveld, F., WO89/01517, which are hereby incorporated by reference in their entirety. However, the application of these same methods to the production of .delta.-globin have been unsuccessful.
Prior to the present application, there have been studies directed towards understanding the reasons for the poor production of the .delta.-globin chain compared to .beta.-globin. Wood et al., 1978, CELL 15:437, found that there were substantial differences between .beta.- and .delta.-globin at all stages of the synthetic process. Those workers reported that the rates of synthesis and of processing of the primary .delta.-globin transcripts were less than that of .beta.-globin; that the life-time of the mature transcript was shorter and that the rate of translation of .delta.-globin was less than that of .beta.-globin. One explanation of the reason for these differences may be the particular nucleotide sequences (codons) used to encode each amino acid. Spritz, R. A., et al., 1980, CELL 21:639, reports that at least two of the codons used in human .delta.-globin are not used in other globins.
More recently, LaFlamme et al., 1987, J.Biol.Chem. 262:4819, considered the production of human .delta.-globin mRNA in a transfected mouse erythroleukemia cell line, which was induced to produce quantities of globins. LaFlamme's studies concerned the effects on the production of .delta.-globin mRNA of the intervening sequences, the portions of the gene between the sequences that encode the globin protein. Each globin gene has two regions of intervening sequences (IVS), sequences that are removed from the primary transcript also known as introns. LaFlamme et al. exchanged the longer IVSs of the .beta. and .delta. genes, called .beta.IVS-2 and .delta.IVS-2, respectively, with the IVSs of the heterologous globin gene. They found that while the introduction of the .delta.IVS-2 into the .beta.-globin gene had a significant negative effect on the production of .beta.-globin mRNA, the substitution of .beta.IVS-2 into the .delta.-globin gene did not increase .delta.-globin mRNA production to any appreciable extent.